This invention relates to proteolytic enzyme compositions which are substantially free of amylase activity, and to a method for preparing such compositions. In particular, this invention relates to proteolytic enzyme compositions which are prepared by a unique method for differentially or selectively inactivating amylase in mixtures of protease and amylase.
Proteolytic enzyme compositions are derived by well known techniques from bacterial and fungal cultures as well as animal organs such as the pancreas. In particlar, the production of protease-containing enzyme mixtures from bacterial sources is well known. Usually, amylase enzymes are co-produced during the growth of the microorganism. For certain purposes, it is desired to provide a protease-containing preparation which is substantially free of any amylase enzyme. For example, applications which require enzymic action on the protein phase of a substance without affecting its amylase content would desirably employ a protease enzyme preparation free of amylolytic activity. Thus, in the treatment of soy flour with protease, for certain purposes it is desirable to use an amylase-free enzyme preparation.
Various known methods of purifying and isolating enzymes include fractional precipitation with inorganic salts such as sodium and ammonium sulfates or polymeric precipitants; organic solvents such as alcohols and ketones; ion exchange chromatography; selective absorption and elution with calcium phosphate gels; separation on columns of CMC or DEAE cellulose; Sephadex gel filtration; differential heat inactivation at varying pH's; isoelectric precipitation; ultrafiltration; and ultracentrifrifugation. Further description of conventional techniques for enzyme purification and isolation is found in Process Biochemistry, August 1973, page 9 et seq. and references cited therein.
A particularly efficacious method for removing undesirable enzyme activity from crude compositions comprises treating the composition to inactivate the contaminant enzyme while leaving the desired activity substantially intact. Hence, although the inactive contaminant is present in the composition it is as effectively neutralized as if it has been physically removed. Inactivation in this context is an irreversible inactivation rather than mere inhibition. Thus, upon removal or dilution of the inactivating agent there is no reappearance of the inactivated enzyme activity. The cost of these inactivation methods is considerably less than purification techniques which requires expensive reagents and multiple processing and separation steps. Such an inactivation method is disclosed in U.S. Pat. No. 2,683,682, where the pH of a proteinase and alpha-amylase preparation is adjusted to differentially inactivate either proteinase or alpha-amylase.
With the foregoing prior art in mind, it is an object of this invention to provide proteolytic enzyme compositions which have been rendered substantially free of amylase activity without the need for lengthy separatory steps or the use of expensive reagents.
It is a further object of this invention to provide a method for treating mixtures of proteolytic and amylolytic enzymes, particularly those derived from bacterial and animal sources, to obtain proteolytic enzyme compositions which are substantially free of amylase activity.
It is an additional object of this invention to provide a method for inactivating amylase in protease compositions while simultaneously destroying any microorganisms present.
It is a further object of this invention to provide proteolytic enzyme compositions substantially free of amylase activity which are stable upon storage.
These and other objects of the invention will be apparent to those skilled in the art from a consideration of this specification taken in its entirety.